RESUMO
This work presents the design of a new protein based on the adenosine triphosphate-binding cassette (ABC) transporter solute binding protein (SBP) derived from Agrobacterium vitis, a gram-negative plant pathogen. The Protein Data Bank in Europe's dictionary of chemical components was utilized to identify sorbitol and D-allitol. Allitol bound to an ABC transporter SBP was identified in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB). Wizard Pair Fitting and Sculpting tools in PyMOL were used to replace bound allitol with sorbitol. PackMover Python code was used to induce mutations in the ABC transporter SBP's binding pocket, and changes in free energy for each protein-sorbitol complex were identified. The results indicate that adding charged side chains forms polar bonds with sorbitol in the binding pocket, thus increasing its stabilization. In theory, the novel protein can be used as a molecular sponge to remove sorbitol from tissue and therefore treat conditions affected by sorbitol dehydrogenase deficiency.
Assuntos
Proteínas de Bactérias , Sorbitol , Proteínas de Bactérias/metabolismo , Transportadores de Cassetes de Ligação de ATPRESUMO
Extracellular vesicles (EV) are important mediators of cell communication and physiology. EVs are frequently investigated by transiently transfecting cells with plasmid DNA to produce EVs modified with protein(s) or nucleic acid(s) of interest. DNA-transfection reagent complexes (DTC) are approximately the same size as EVs, raising the possibility that some purification procedures may fail to separate these two species and activity arising from carryover DTC may be improperly attributed to EVs. We find that differential ultracentrifugation, a commonly employed EV isolation procedure, does not separate EVs from DTC present in the cell culture supernatant of transiently transfected cells. We demonstrate that the biological activity of an EV-directed Cre recombinase is due to contaminating plasmid DNA and not EV-mediated delivery of Cre protein. Moreover, steps commonly taken to remove plasmid DNA from EV samples, such as media exchanges and treatment with nucleases, are ineffective at avoiding this artefact. Due to the pernicious nature of plasmid DNA in these cellular assays, some reports of EV function are likely artefacts produced by contaminating DTC. EVs and DTC can be separated by density gradient ultracentrifugation, highlighting the importance of validating elimination of DTC when using transient transfection of EV-producing cells to interrogate EV function.
Assuntos
Vesículas Extracelulares , Ácidos Nucleicos , Artefatos , DNA/metabolismo , Vesículas Extracelulares/genética , Indicadores e Reagentes/metabolismo , Ácidos Nucleicos/metabolismo , TransfecçãoRESUMO
The transient period of regeneration potential in the postnatal heart suggests molecular changes with maturation influence the cardiac response to damage. We have previously demonstrated that injury and exercise can stimulate cardiomyocyte proliferation in the adult heart suggesting a sensitivity to exogenous signals. Here, we consider whether exogenous fetal ECM and mechanically unloading interstitial matrix can drive regeneration after myocardial infarction (MI) surgery in low-regenerative hearts of day5 mice. Compared to controls, exogenous fetal ECM increases cardiac function and lowers fibrosis at 3 weeks post-injury and this effect can be augmented by softening heart tissue. In vitro experiments support a mechano-sensitivity to exogenous ECM signaling. We tested potential mechanisms and observed that fetal ECM increases nuclear YAP localization which could be enhanced by pharmacological stabilization of the cytoskeleton. Blocking YAP expression lowered fetal ECM effects though not completely. Lastly we observed mechanically unloading heart interstitial matrix increased agrin expression, an extracellular node in the YAP signaling pathway. Collectively, these data support a combined effect of exogenous factors and mechanical activity in altering agrin expression, cytoskeletal remodeling, and YAP signaling in driving cardiomyocyte cell cycle activity and regeneration in postnatal non-regenerative mice. STATEMENT OF SIGNIFICANCE: With the purpose of developing regenerative strategies, we investigate the influence of the local niche on the cardiac injury response. We conclude tissue stiffness, as anticipated in aging or disease, impairs regenerative therapeutics. Most novel, mechanical unloading facilitates enhanced cardiac regeneration only after cells are pushed into a permissive state by fetal biomolecules. Specifically, mechanical unloading appears to increase extracellular agrin expression that amplifies fetal-stimulation of nuclear YAP signaling which correlates with observed increases of cell cycle activity in cardiomyocytes. The results further suggest the cytoskeleton is critical to this interaction between mechanical unloading and independently actived YAP signaling. Using animal models, tissue explants, and cells, this work indicates that local mechanical stimuli can augment proliferating-permissive cardiomyocytes in the natural cardiac niche.